Activin receptor type 2A (ACVR2A) functions directly in osteoblasts as a negative regulator of bone mass

Abstract

Bone and skeletal muscle mass are highly correlated in mammals, suggesting the existence of common anabolic signaling networks that coordinate the development of these two anatomically adjacent tissues. The activin signaling pathway is an attractive candidate to fulfill such a role. Here, we generated mice with conditional deletion of activin receptor (ACVR) type 2A, ACVR2B, or both, in osteoblasts, to determine the contribution of activin receptor signaling in regulating bone mass. Immunohistochemistry localized ACVR2A and ACVR2B to osteoblasts and osteocytes. Primary osteoblasts expressed activin signaling components, including ACVR2A, ACVR2B, and ACVR1B (ALK4) and demonstrated increased levels of phosphorylated Smad2/3 upon exposure to activin ligands. Osteoblasts lacking ACVR2B did not show significant changes in vitro However, osteoblasts deficient in ACVR2A exhibited enhanced differentiation indicated by alkaline phosphatase activity, mineral deposition, and transcriptional expression of osterix, osteocalcin, and dentin matrix acidic phosphoprotein 1. To investigate activin signaling in osteoblasts in vivo, we analyzed the skeletal phenotypes of mice lacking these receptors in osteoblasts and osteocytes (osteocalcin-Cre). Similar to the lack of effect in vitro, ACVR2B-deficient mice demonstrated no significant change in any bone parameter. By contrast, mice lacking ACVR2A had significantly increased femoral trabecular bone volume at 6 weeks of age. Moreover, mutant mice lacking both ACVR2A and ACVR2B demonstrated sustained increases in trabecular bone volume, similar to those in ACVR2A single mutants, at 6 and 12 weeks of age. Taken together, these results indicate that activin receptor signaling, predominantly through ACVR2A, directly and negatively regulates bone mass in osteoblasts.

Keywords: activin; bone; myostatin; osteoblast; transforming growth factor beta (TGF-β).

Figure 1.

Soluble activin receptor administration increases bone volume in vivo and enhances osteoblast differentiation in vitro. ACVR2A/Fc and ACVR2B/Fc treatment demonstrates significant increases in skeletal muscle weights as compared with vehicle-treated controls (A). ACVR2A/Fc administration exhibits a near doubling in trabecular bone volume/tissue volume and a significant increase in cortical thickness (B). Similarly, ACVR2B/Fc treatment demonstrates a near tripling in trabecular bone volume/tissue volume but no significant increase in cortical thickness (C). Both ACVR2A/Fc and ACVR2B/Fc treatment demonstrate significant increases in calvarial bone volume (D). ACVR2B/Fc treatment shows a dose-dependent reduction of Smad2 phosphorylation in vitro (E). Further in vitro analysis demonstrates that ACVR2B/Fc treatment enhances alkaline phosphatase activity and mineral deposition by Alizarin Red staining in osteoblast differentiation cultures (F). *, p < 0.05.

Figure 2.

Activin receptor signaling components are expressed and functional in osteoblasts. Transcriptional expression assays demonstrate that the activin signaling components are expressed in primary osteoblasts as compared with skeletal muscle (A). ACVR2A and ACVR2B increase transcriptional expression with osteoblast differentiation (B). Immunohistochemistry localizes ACVR2A (white arrowheads, left) and ACVR2B (white arrowheads, right) to osteoblasts and osteocytes in bone sections (C). Phosphorylation of Smad2 following activin ligand administration demonstrates activin receptor functionality in osteoblasts (D).

Figure 3.

Disruption of ACVR2A enhances osteoblast differentiation in vitro. ΔACVR2A osteoblasts exhibit a slight decrease in proliferation (A), whereas disruption of ACVR2B does not alter osteoblast proliferation (B). Osteoblast differentiation is significantly enhanced with ACVR2A disruption as shown by increased alkaline phosphatase activity (C) and mineral deposition (D). ACVR2B disruption does not affect alkaline phosphatase activity (E) or mineral deposition (F). Disruption of ACVR2A induces increased transcriptional expression of osteoblast differentiation markers such as Osterix, Osteocalcin, and Dmp1 at day 7 (G). *, p < 0.05.

Figure 4.

Femurs of ΔACVR2A male mice exhibit increased bone volume. Allele-specific PCR demonstrates ACVR2A (A) and ACVR2B (B) recombination exclusively at skeletal sites. Femurs from ΔACVR2A mice exhibit increases in trabecular bone parameters (C–F), including bone volume/tissue volume (C) and trabecular number (E) at 6 weeks of age. Femurs of ΔACVR2A mice at 12 weeks of age demonstrate increases in trabecular bone volume/tissue volume (C), trabecular thickness (D), trabecular number (E), and a decrease in trabecular spacing (F). Femoral cortical parameters (G–J) were unchanged at 6 weeks of age in ΔACVR2A mice. However, cortical tissue area (G), bone area (H), and cross-sectional thickness (J) were increased, whereas cortical bone area/tissue area (I) was unchanged at 6 and 12 weeks of age. *, p < 0.05; **, p < 0.005.

Figure 5.

ACVR2B disruption does not affect bone volume in vivo. Femurs of ΔACVR2B male mice show no significant changes in trabecular (A–D) or cortical bone parameters (E–H) at 6 or 12 weeks of age. Trabecular bone parameters include bone volume/tissue volume (A), trabecular thickness (B), trabecular number (C), and trabecular spacing (D). Apart from a slight reduction in bone area/tissue area at 6 weeks of age (G), there are no significant changes observed in cortical parameters, including tissue area (E), bone area (F), bone area/tissue area (G), and cross-sectional thickness (H) in 6- or 12-week-old ΔACVR2B male mice. *, p < 0.05.

Figure 6.

Femurs of ΔACVR2A/2B mice exhibit similar changes in bone volume as ΔACVR2A mice. Femurs of ΔACVR2A/2B male mice exhibit similar increases in trabecular bone parameters (A–D) as ΔACVR2A mice. ΔACVR2A/2B mice demonstrate increases in trabecular bone volume/tissue volume (A), trabecular number (C), and a decrease in trabecular spacing (D) at 6 weeks of ago. These increases were sustained in trabecular bone volume/tissue volume (A) and trabecular number (C) at 12 weeks of age. However, no significant changes were seen in trabecular thickness (B) at 6 or 12 weeks of age. Cortical bone parameters, including tissue area (E), bone area (F), bone area/tissue area (G), and cross-sectional thickness (H), were unchanged in 6- and 12-week-old ΔACVR2A/2B male mice. *, p < 0.05.

Figure 7.

Skeletal changes in ΔACVR2A mice are accompanied by increased mechanical properties. Three-point bending analysis of femurs from ΔACVR2A mice demonstrates significant increases in mechanical properties, such as the ultimate moment (A), bending rigidity (B), pre-yield strain (E), pre-yield energy (F), and pre-yield toughness (G), and nonsignificant trends in ultimate stress (C). There were, however, no significant changes in the ultimate bending energy (D) or Young's modulus (H) with ACVR2A disruption. *, p < 0.05.

Figure 8.

Activin receptor signaling is a significant contributor to anabolic changes observed with soluble receptor administration. Soluble activin receptor treatment with ACVR2B/Fc demonstrates attenuated increases in bone volume/tissue volume (A), trabecular thickness (B), and trabecular number (C) in ΔACVR2A/2B mice. There were no differential changes in trabecular bone spacing (D) or differential increases in skeletal muscle weights, including the gastrocnemius (E), tibialis anterior (F), or quadriceps (G) muscles. *, p < 0.05.